Enteroviruses are transmitted by the fecal-oral route, and must cross the intestinal mucosa to initiate infection. The intestines are lined by polarized epithelial cells, with distinct apical and basolateral surfaces; intercellular tight junctions prevent virus access to receptors on basolateral membranes. Many enteroviruses, including many coxsackie B viruses (CBV) and echoviruses (EV), appear to have evolved independently to bind to decay accelerating factor (DAF), a molecule expressed on the surface of polarized epithelium. We believe that these viruses use DAF to interact with epithelial cells in the intestinal lumen. DAF does more than simply provide a site for virus attachment. We recently found that it initiates at least two intracellular signaling pathways required for CBV entry and infection in polarized cells: activation of Abl kinase leads to actin rearangements and virus movement to the tight junction, where interaction with the coxsackievirus and adenovirus receptor (CAR) initiates the uncoating process that releases viral RNA; activation of Fyn kinase triggers virus internalization in caveolar vesicles, and delivery to the endoplasmic reticulum (ER) where uncoating is completed before replication ensues. We have more recently determined that virus entry from the tight junction is linked to the internalization of a tight junction protein, occludin, and that both virus entry and occludin internalization occur by a process that combines aspects of caveolar endocytosis with features characterisitc of macropinocytosis. These results suggest that infection of polarized cells is a complex process likely to involve distinctive cell biological mechanisms. We propose three sets of experiments to examine the cell biology of infection in polarized epithelium. 1. We will define the pathway by which DAF-binding EV enter polarized epithelium, following the entry of labeled virus, and using specific inhibitors (drugs, siRNAs) to dissect the underlying cellular processes. 2. We will determine the function of actin rearrangements and changes in microvillus architecture during virus entry, and identify the mechanism by which actin reorganization is initiated in response to virus-DAF interaction. 3. We will screen a siRNA library to identify signaling molecules required for CBV entry into polarized epithelium, and define the role of specific signaling molecules in the process of infection. PUBLIC HEALTH RELEVANCE: Enteroviruses are the major cause of viral meningitis, and the second major cause of myocarditis in the US, and interaction with polarized epithelium is the first step in infection. Understanding the special mechanisms by which enteroviruses infect polarized cells will reveal new potential drug targets.